Alphaviruses are divided into distinct, geographically isolated groups, the New World (NW) and Old World (OW) alphaviruses. Different alphavirus species display adaptations specific to particular hosts and mosquito vectors and induce diverse diseases in humans and other mammals. These adaptations are driven by the evolution of viral structural genes, which has resulted in the formation of six major alphavirus serocomplexes. Evolution of the nonstructural proteins nsP1, nsP2 and nsP4 is restricted by their enzymatic activities in RNA replication, which have been well characterized. However, functions of nsP3 in alphavirus biology, besides knowing that this protein is an essential component of replication complexes, are less understood. The amino- terminal fragment of nsP3 demonstrates a high level of conservation between alphavirus species. The carboxy-terminal domain is highly phosphorylated, exhibits a very low level of sequence identity between alphaviruses, and is thusly referred to as the hypervariable domain or HVD. Our recent NMR studies have demonstrated that nsP3-specific HVD of chikungunya virus (CHIKV) is disordered. It contains linear motifs that interact with a distinct set of protein factors, and these interactions are indispensable for CHIKV replication. Most of the interacting host factors are represented by protein families, where each member is capable of supporting viral RNA replication. Another level of redundancy is achieved by the abilities of some of the interacting protein families to independently mediate viral replication complex formation. Our central hypothesis is that cellular nsP3 HVD-binding proteins mediate assembly of CHIKV replication complexes. In the proposed research, we intend to investigate the mechanism of CHIKV HVD interactions with cellular SH3 domain- containing proteins, with the main focus on the CD2AP protein. This interaction plays a pro-viral role in CHIKV replication in vertebrate cells and is more critical for viral replication in mosquito cells. By using a variety of biochemical and NMR-based approaches, we will precisely define binding sites for BIN1, CD2AP and SH3KBP1 in CHIKV HVD, analyze localization of BIN1-, CD2AP- and SH3KBP1-HVD complexes in the infected cells and define the role of SH3 domain containing proteins in different steps of CHIKV replication. Our future studies will result in development of new means of therapeutic treatment and attenuated vaccine candidates that cannot be transmitted by mosquitoes.